Low dose oral tolerance is defined as the active suppression of a systemic immune response to a specific antigen by regulatory T cells (Tregs) generated in response to repeated ingestion of that antigen. Though ex- tensively studied, the specific mechanisms involved in the generation of regulatory cells in response to oral antigen remain poorly understood. Although previous studies have demonstrated that oral antigen can in- duce both CD4+ and CD8+ Tregs, the role of CD8+ T cells remains controversial. Our preliminary data dem- onstrate that feeding C57BL/6 mice with the MHC Class'I immunodominant peptide of OVA (SIINFEKL) generates tolerance to subsequent immunization with the whole OVA protein, and that this response is de- pendent on the presence of CD8+ T cells. Our hypothesis is that CD8+ T cells are a major component of the tolerogenic response to oral antigen in a normal immune setting. SIINFEKL binds directly to MHC Class I molecules and therefore stimulates only CD8+ T cells. We propose to harness this to assess the role of CD8+ Tregs in oral tolerance, independently of CD4+ T cells, using an intact immune system. In specific aim 1, we will confirm that SIINFEKL feeding induces the generation of CD8+ Tregs by transfer of tolerance to na- ive mice with CDS" T cells isolated from antigen fed mice, and then localize the sites of antigen uptake and presentation by feeding fluorescent-labeled peptide or protein. In specific aim 2, we will establish the mechanism of how CD8+ Tregs induce suppression, by first establishing whether the mechanism of suppres- sion requires cognate or noncognate interactions using in-vitro suppression assays. We will then identify the mediators involved using flow cytometry by staining SIINFEKL-specific cells with antigen-loaded pentamer, and costaining with antibody against different combinations of phenotypic markers for CD8+ T cell function or regulatory function. Finally, in specific aim 3, we will establish the importance of high affinity interactions be- tween TCR and peptide-MHC complexes in the induction of CD8+ Tregs by feeding peptide variants of SIINFEKL. This is of particular importance as this is the first study to investigate the importance of TCR- antigen affinity in the generation of Tregs in oral tolerance. While numerous studies have demonstrated the therapeutic potential of low dose oral tolerance in the treatment of various animal models of autoimmune disease, several clinical trials have found it to be only marginally successful in the amelioration of disease in humans. The results of these trials have demonstrated that while oral administration of antigen can influ- ence systemic immune responses, there is a need for a more complete understanding of the mechanisms underlying the induction of oral tolerance in order to optimize treatment protocols.